This invention pertains generally to the field of devices and techniques for microbial growth assays and to the screening of substances for their effect on cell growth.
Several types of microbial growth assays have been developed, some with commercial success, but most of these assays rely on flow-through chambers, large (greater than ml) sample sizes, or long incubation periods, which limits the suitability of such assays for high-throughput screening. Targeted against a range of wild-type or enfeebled strains of various pathogenic bacteria, the growth assay is the simplest and most direct measurement of drug efficacy, and represents the means by which almost all antimicrobial drugs have been discovered. The classic culture methods used by Fleming for the discovery of penicillin are still applied in clinical diagnostics today, and have been the primary path for drug discovery as well, even though response times are typically measured in days. Faster techniques that have been developed for measuring cell concentrations still employ relatively large volumes. These techniques and devices include optical measures of turbidity, flow cytometers, biomass measurements with microbalances, and electronic counting techniques, predominantly the Coulter counter. These techniques can still take hours to respond, given the large populations being measured.
Because of the small size of most bacteria, conventional optical measurements of scattering or attenuation lack the combination of speed and selectivity needed for high-density arrays, in part because the dielectric contrast between the cells and their medium is low in the visible portion of the electromagnetic spectrum. High-frequency electrical characterization, however, is attractive for microbial growth assays because it circumvents the need for image analysis and it is readily scalable to manipulating and screening sub-visible particles, such as viruses. See D. W. van der Weide, xe2x80x9cMicroscopes for the sub-visible: scanning the near field in the microwave through infrared,xe2x80x9d Optics and Photonics News, 1998, Vol. 9, pp. 40-45; D. W. E. Allsopp, et al., xe2x80x9cImpedance technique for measuring dielectrophoretic collection of microbiological particles,xe2x80x9d Journal of Physics D (Applied Physics), 1999, Vol. 32(9), pp. 1066-74. Although it operates at low frequencies, the Coulter counter exploits the contrast in conductivity between cells and their medium as the cells traverse an aperture between two chambers: a change in resistance is a discrete event corresponding to the presence of a cell in the aperture. Other more sophisticated high-frequency techniques measure not only the real part (resistance/conductance) but also the imaginary part (reactance/susceptance) of the cells"" impedance/admittance, since cells display a complex permittivity. See R. Pethig, et al., xe2x80x9cThe passive electrical properties of biological systems: their significance in physiology, biophysics and biotechnology,xe2x80x9d Phys Med Biol, 1987, Vol. 32(8), pp. 933-70; A. D. Shaw, et al., xe2x80x9cRapid analysis of high-dimensional bioprocesses using multivariate spectroscopies and advanced chemometrics,xe2x80x9d Adv Biochem Eng Biotechnol, 2000, Vol. 66, pp. 83-113.
A crisis in the management of infectious disease has resulted from the emergence of new pathogens and the development of resistance to old antibiotics. This crisis has generated a great need for improved antibiotic discovery techniques. Some of the potential new sources for antibiotic drugs require the testing of many thousands or millions of samples to find suitable active compounds. Current growth assay technology, which typically requires many hours or days to analyze the effectiveness of a potential new antibiotic, is not well-suited to the economic screening of potential antibiotics on such a massive scale. Reliable, rapid assays which can be carried out in a few hours or less with minimal human intervention are thus urgently needed.
In accordance with the invention, rapid assays of bacterial growth can be carried out in a very short period of time, typically in a few hours or less, to test a wide variety of substances for their effect on bacterial growth. The short time required to determine whether the substances have an effect on bacterial growth coupled with automated measurement of bacterial growth in individual samples allows very high throughput, making feasible the analysis of thousands or tens of thousands of target samples per day. Excellent growth assay accuracy is obtained by obtaining tests with samples of a selected bacterium without an inhibitor of growth, samples in which a known antibiotic is added to the bacteria and growth medium, and samples in which no bacterium is present. Only very small amounts of the substances to be tested are required for the test, minimizing the costs and allowing the evaluation of substances that are in short supply.
The microbial growth assay apparatus of the invention includes microbial growth assay wells, which may be provided in a base plate having a top surface, each well preferably having a liquid capacity of 30 microliters (xcexcl) or less or being filled with fluid contents of 30 xcexcl or less and preferably in the range of a few tens of nanoliters. Electrodes are coupled together through the wells, and electrical connectors are connected to the electrodes, to enable electrical measurements of the impedance of the content of each well. For example, the effect of the content of the wells may be measured by measuring the capacitance between the electrodes at each well and/or the electrodes may be formed to make conductive contact with the contents of the wells to allow measurements of the electrical resistance of the contents. The electrical measurements of the contents of each well are made over a period of time during which a cell population in the growth medium in the well may be expected to rapidly increase to provide a measurable change in the capacitance or other electrical properties measured at the well. Because of the small amount of liquid content in the wells, a small number of bacteria introduced into each well (e.g., 50-100) will rapidly multiply to a size sufficient to saturate the well in a short period of time, a few hours or less, particularly with a common bacterium such as E. coli. 
To carry out multiple sample tests at a high density, a base plate is utilized which has a top surface and a plurality of microbial growth assay wells formed as depressions in the base plate which extend below the top surface, with the wells arranged in a rectangular matrix pattern, each of the wells having a liquid capacity of 30 xcexcl or less and preferably in the range of tens of nanoliters. A first electrode for each well may be formed under the well and a second electrode for each well may be formed on a cover mounted over the base plate such that a capacitor is formed between the first and second electrodes for each well with the contents of the well between the electrodes, allowing the effect of the contents of the wells to be measured by measuring the capacitance between the electrodes at each well. The electrodes may also be formed coaxially, with an inner electrode and a coaxial outer electrode. The apparatus may further include a meter that measures capacitance and a switching unit, the switching unit electrically connected to the meter and electrically connected individually to the electrodes for each of the plurality of wells, with the switching unit switchable to selectively connect the electrodes for one of the wells at a time to the meter. The meter can measure the capacitance between the electrodes and also preferably can measure conductance and resistance between the electrodes, as appropriate. A computer may be connected to the meter and to the switching unit to provide control signals to the switching unit and the meter to control the connection of the electrodes from individual wells to the meter and to receive a signal from the meter for each well that is coordinated with the switching of the switching unit to connect the electrodes for the well to the meter. The wells may be formed in small sizes, e.g., few microns on a side, with a preferred liquid capacity range of a few tens of nanoliters, and arranged in very high density on a standard microtiter size base plate, e.g., with a well density from several hundred to several thousand wells per base plate, facilitating the rapid and simultaneous testing of a large number of different substances for their effects on bacterial growth.